Porcine small intestinal submucosal (SIS) is being used successfully as a xenograft in several species without any evidence of adverse immunologic reactions. Our preliminary data suggest that there is an immune response to SIS but it is restricted to the T helper type 2 (Th2) pathway which is characterized by increased expression of IL-10 and production of serum IgG1 anti-SIS antibody. We will now investigate in detail the basis for SIS accommodation, the role of anti-inflammatory cytokines in preventing rejection, and the properties of naturally- occurring anti-SIS antibodies in normal serum. Graft tissue and spleens will be tested for cytokine expression by reverse transcriptase-PCR and ribonuclease protection analysis at various times after SIS implantation in mice. Serum antibody levels will be quantitated by isotype-specific ELISAs. Effects of SIS on murine responses to pathogenic microorganisms will be tested by vaccination with attenuated influenza virus and pneumococcal polysaccharide vaccine, and measuring specific antibody titers by ELISA and protection from disease by challenge with infectious organisms. The hypothesis that inhibition of Th1 cell stimulation is responsible for preventing rejection of SIS skin grafts will be assessed using mice with a specific disruption in the IL-10 gene and the potential importance of transforming growth factor-beta will be determined by in vivo antibody-mediated neutralization. Rejection of SIS grafts will also be tested in mice injected with IL-12 which is a potent inducer of the Th1 cell pathway. Finally, the presence of anti- SIS antibodies in normal human serum will be determined by immunocytochemical staining of SIS-coated slides. Specific galactose (Gal)-containing inhibitors will be used during staining to establish antigen specificity and the bound antibodies will be assessed for relative expression of individual isotypes. The ability of anti-SIS antibodies to activate complement will be tested by measuring generation of C3a and cytotoxicity for co-cultured cells will be determined using human endothelial cells which lack Gal epitopes. The results will provide important insight into the mechanisms responsible for successful xenogeneic transplantation and could lead to targeted cytokine therapies to prevent tissue rejection.